Dataset Information

The ryanodine receptor agonist 4-chloro-3-ethylphenol blocks ORAI store-operated channels.

ABSTRACT: Depletion of the Ca(2+) store by ryanodine receptor (RyR) agonists induces store-operated Ca(2+) entry (SOCE). 4-Chloro-3-ethylphenol (4-CEP) and 4-chloro-m-cresol (4-CmC) are RyR agonists commonly used as research tools and diagnostic reagents for malignant hyperthermia. Here, we investigated the effects of 4-CEP and its analogues on SOCE.SOCE and ORAI1-3 currents were recorded by Ca(2+) imaging and whole-cell patch recordings in rat L6 myoblasts and in HEK293 cells overexpressing STIM1/ORAI1-3.4-CEP induced a significant release of Ca(2+) in rat L6 myoblasts, but inhibited SOCE. The inhibitory effect was concentration-dependent and more potent than its analogues 4-CmC and 4-chlorophenol (4-ClP). In the HEK293 T-REx cells overexpressing STIM1/ORAI1-3, 4-CEP inhibited the ORAI1, ORAI2 and ORAI3 currents evoked by thapsigargin. The 2-APB-induced ORAI3 current was also blocked by 4-CEP. This inhibitory effect was reversible and independent of the Ca(2+) release. The two analogues, 4-CmC and 4-ClP, also inhibited the ORAI1-3 channels. Excised patch and intracellular application of 4-CEP demonstrated that the action site was located extracellularly. Moreover, 4-CEP evoked STIM1 translocation and subplasmalemmal clustering through its Ca(2+) store-depleting effect via the activation of RyR, but no effect on STIM1 redistribution was observed in cells co-expressing STIM1/ORAI1-3.4-CEP not only acts as a RyR agonist to deplete the Ca(2+) store and trigger STIM1 subplasmalemmal translocation and clustering, but also directly inhibits ORAI1-3 channels. These findings demonstrate a novel pharmacological property for the chlorophenol derivatives that act as RyR agonists.

SUBMITTER: Zeng B

PROVIDER: S-EPMC3952802 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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The ryanodine receptor agonist 4-chloro-3-ethylphenol blocks ORAI store-operated channels.

Zeng Bo B Chen Gui-Lan GL Daskoulidou Nikoleta N Xu Shang-Zhong SZ

British journal of pharmacology 20140301 5

<h4>Background</h4>Depletion of the Ca(2+) store by ryanodine receptor (RyR) agonists induces store-operated Ca(2+) entry (SOCE). 4-Chloro-3-ethylphenol (4-CEP) and 4-chloro-m-cresol (4-CmC) are RyR agonists commonly used as research tools and diagnostic reagents for malignant hyperthermia. Here, we investigated the effects of 4-CEP and its analogues on SOCE.<h4>Experimental approach</h4>SOCE and ORAI1-3 currents were recorded by Ca(2+) imaging and whole-cell patch recordings in rat L6 myoblasts ...[more]

PMID: 24670147

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Project description:The endoplasmic reticulum (ER) is at the epicenter of astrocyte Ca(2+) signaling. We sought to identify the molecular mechanism underlying store-operated calcium entry that replenishes ER stores in mouse Müller cells. Store depletion, induced through blockade of sequestration transporters in Ca(2+)-free saline, induced synergistic activation of canonical transient receptor potential 1 (TRPC1) and Orai channels. Store-operated TRPC1 channels were identified by their electrophysiological properties, pharmacological blockers, and ablation of the Trpc1 gene. Ca(2+) release-activated currents (ICRAC) were identified by ion permeability, voltage dependence, and sensitivity to selective Orai antagonists Synta66 and GSK7975A. Depletion-evoked calcium influx was initiated at the Müller end-foot and apical process, triggering centrifugal propagation of Ca(2+) waves into the cell body. EM analysis of the end-foot compartment showed high-density ER cisternae that shadow retinal ganglion cell (RGC) somata and axons, protoplasmic astrocytes, vascular endothelial cells, and ER-mitochondrial contacts at the vitreal surface of the end-foot. The mouse retina expresses transcripts encoding both Stim and all known Orai genes; Müller glia predominantly express stromal interacting molecule 1 (STIM1), whereas STIM2 is mainly confined to the outer plexiform and RGC layers. Elimination of TRPC1 facilitated Müller gliosis induced by the elevation of intraocular pressure, suggesting that TRPC channels might play a neuroprotective role during mechanical stress. By characterizing the properties of store-operated signaling pathways in Müller cells, these studies expand the current knowledge about the functional roles these cells play in retinal physiology and pathology while also providing further evidence for the complexity of calcium signaling mechanisms in CNS astroglia.Significance statementStore-operated Ca(2+) signaling represents a major signaling pathway and source of cytosolic Ca(2+) in astrocytes. Here, we show that the store-operated response in Müller cells, radial glia that perform key structural, signaling, osmoregulatory, and mechanosensory functions within the retina, is mediated through synergistic activation of transient receptor potential and Orai channels. The end-foot disproportionately expresses the depletion sensor stromal interacting molecule 1, which contains an extraordinarily high density of endoplasmic reticulum cisternae that shadow neuronal, astrocytic, vascular, and axonal structures; interface with mitochondria; but also originate store-operated Ca(2+) entry-induced transcellular Ca(2+) waves that propagate glial excitation into the proximal retina. These results identify a molecular mechanism that underlies complex interactions between the plasma membrane and calcium stores, and contributes to astroglial function, regulation, and response to mechanical stress.

Dataset Information

The ryanodine receptor agonist 4-chloro-3-ethylphenol blocks ORAI store-operated channels.

Publications

The ryanodine receptor agonist 4-chloro-3-ethylphenol blocks ORAI store-operated channels.

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